Non-muscle-invasive bladder cancer has few available treatments. The mainstay of treatment is surgery with a transurethral resection of the bladder tumor but the recurrence rate is as high as 75%. Mitomycin C is given peri-operatively to reduce the recurrence rate and when disease is high grade, Bacillus Calmette-Guerin (BCG) is used to prevent both recurrence and progression. However, these treatments often fail and when successful, they work through a poorly understood mechanism. Therefore, attention has been directed towards molecular alterations in bladder cancer to identify novel targets. Mutation analysis of 145 urothelial tumors found that FGFR3 and PIK3CA mutations were most commonly found in low grade non-muscle-invasive tumor (Sjodahl et al, 2011). In fact, the FGFR3 mutation is found in up to 80% in low-grade bladder tumors and up to 50% of invasive tumors. In addition, over-expression or amplification of EGFR is seen in the majority of urothelial tumors (Rotterud et al, 2005). However, targeted therapy with the monoclonal antibodies has been disappointing in bladder cancer. Multiple studies have been conducted with EGFR-targeted monoclonal antibodies and tyrosine kinase inhibitors without any significant clinical benefit despite encouraging pre-clinical results (Black et al, 2012). Recently, an FGFR3 monoclonal antibody was effective but primarily in invasive bladder cancer cell lines instead of the low grade bladder cancer where mutations in FGFR3 are more commonly found (Qing et al, 2009). Despite having these well-defined targets in non-muscle-invasive bladder cancer, there has been little success with targeted therapy alone. Recently, colleagues from the molecular imaging branch demonstrated successfully that humanized monoclonal antibodies could be used to target infrared light activated compounds selectively into colon cancer cells. Upon introduction of infrared light, these compounds become activated to induce cell death (Mitsunaga et al, 2011). These antibody-infrared light activated drug conjugates are able to capitalize on the targetable property of the antibodies but rely on the cytotoxicity of the drug conjugated to the antibody and not the antibody itself. Given the prevalence of EGFR and FGFR3 mutations in non-muscle-invasive bladder cancer and the disappointing results with standard targeted therapy, we postulate that antibody-infrared light activated drug conjugates will have significant activity in inducing cell death selectively in bladder cancer cells. This is novel as an infrared light could be attached to existing urologic equipment such as a urinary catheter or flexible cystoscope and be introduced into the bladder to activate such conjugates instilled into the bladder should pre-clinical work suggest a benefit of these agents in bladder cancer. We will further investigate molecular targeted photoimmunotherapy in bladder cancer by specifically targeting EGFR, FGFR3, and other related targets. The objectives are: 1) To profile bladder cancer cell lines by surface receptor expression and mutations so as to better understand their role as models of urothelial cancer; 2) to establish in vitro efficacy of photodynamic therapy (specifically photoimmunotherapy); 3) to determine the mechanism of cell death caused by photodynamic therapy (PIT); 4) to use antibody-drug conjugates in animals and humans alone and in synergistic combinations; 5) evaluate for immunogenic cell death. Our key accomplishments are as follows: 1) we have obtained a large number of bladder cancer cell lines and we have personally developed three cell lines within the Urologic Oncology Branch from patient tumors. These cell lines have been thoroughly characterized using a mutational array. Furthermore, several cell lines have been successfully transfected with luciferase and we have begun orthotopic implantation with these lines; 2) we have thoroughly profiled several bladder cancer cell lines for surface expression of growth factor receptors for which monoclonal antibodies are available. We are now embarking on characterizing them for FGFR3 expression; 3) we have confirmed the efficacy of photoimmunotherapy (aim 2) and determined that necrosis is the likely mechanism of action (aim 3). We have completed animal studies (aim 4) and confirmed presence of EGFR and other targets in actual human tumors by quantitative immunofluorescence. We published our first manuscript describing our in vitro and in vivo work in Molecular Cancer Therapeutics. We just completed combination antibody-drug conjugates using EGFR and HER-2 targeted approach concurrently (aim 4) and are submitting the results for publication. In this upcoming report, we have shown that combination of two antibody-drug conjugates can be helpful in tumors with lower expression of each individual target. We also have a review article on photodynamic therapy and photoimmunotherapy in press. We are now trying to establish that photoimmunotherapy results in immunogenic cell death which can prime the immune system (aim 5). After confirming our findings in an immunocompetent mouse, we will combine photoimmunotherapy (single antibody-drug conjugate and multiple antibody-drug conjugates) with immunotherapy (PD-1/PD-L1 therapy - aim; 4). Finally, we hope to partner with the NCI or a commercial collaborator to introduce such therapy to patients in a phase I clinical trial.